This grant concerns unusual proteins that have the capacity to change shape and to selectively template that change in shape to other proteins of the same type, a property shared by prions and other amyloid-forming proteins across the evolutionary spectrum. Amyloids have devastating personal and economic costs in an extraordinary variety of settings, many of which have only become clear in recent years. These range from degenerative diseases of our aging population to the production of infectious bacterial biofilms that resist eradication by antibiotics, bacteriophage, and even bleach. This project takes advantage of the remarkable methodological approaches yeast cells offer for the study of difficult problems in protein folding and assembly. It also capitalizes on technological innovations recently developed in our laboratory and others for structural and mechanical investigations of amyloid proteins. The model protein for much of this work is Sup35, a translation termination factor of yeast. It has provided a wealth of information on nucleation and assembly pathways in vitro. Also strains of this prion with defined phenotypic consequences and patterns of inheritance have been intensely investigated in vivo. In recent years dozens of new yeast prions have been discovered by our lab and others. By focusing on this class of proteins, proteins with completely unrelated amino acid sequences and diverse biological functions, we aim to uncover fundamental insights on the assembly of these proteins that will be applicable to a variety of problems that currently cause great suffering and economic hardship. Our aims include i) Obtaining an atomic-level structural understanding of the amyloid fibers formed the prion domain of model prion Sup35 (called NM), using solid-state NMR; (ii) Characterizing the mechanical properties of Sup35 prion fibers using optical trapping and microfluidic techniques; (iii) Employing fluorescence microscopy techniques to monitor the dynamic processes involved in prion replication and inheritance in living cells. This work will provide fundamental new knowledge with potential applications to both normal biology and disease.